PROJECT SUMMARY The completion of the human genome was supposed to provide a definitive list of all protein-coding genes in cells and tissues; however, the recent discovery of hundreds, potentially thousands, of novel protein-coding genes has revealed an unexpected blind spot in gene annotation for small protein-coding open reading frames (smORFs). The goals of this application are to 1) annotated all human and mouse smORFs (i.e., the smORFeome) (Aim 1), and 2) characterize the biochemical (Aim 2) and cellular (Aim 3) functions of selected smORFs. Achieving these objectives will accomplish the larger goal of defining the protein-coding capacity of the human genome and identifying additional genes with critical functions in biology and disease. Aim 1a utilizes an innovative strategy that integrates next-generation sequencing, bioinformatics, and proteomics to define the human smORFeome in several human cell lines. In Aim 1b, the same approach is applied to mouse tissues to generate the first smORF Tissue Atlas. This Atlas will enable the rapid identification of smORFs with unique patterns of expression or smORFs that are regulated during a disease, thereby accelerating the physiological characterization of these genes. There is extensive overlap between the mouse and human smORFs identified to date, so conclusions from this work should readily extend to human disease. smORF-encoded peptides and small proteins are referred to as microproteins, and in Aims 2a and 2b, two different proteomics strategies will be applied to the identification of microprotein-protein interactions. Microprotein PPIs will reveal the biochemical function(s) of smORFs and accelerate the biological characterization of these genes. Lastly, in Aim 3, experiments will be performed to define the biological role of a smORF with an integral role in the unfolded protein response called PIGBOS. The successful completion of these Aims will increase basic knowledge regarding the protein-coding potential of the human and mouse genomes, and provide functional insights that will lead to improvements in disease diagnosis, treatment, and prevention.